Signal transduction mediated by insulin in nonhematopoietic cells was shown to be mediated by insulin receptor substrate-1 (IRS-1). IRS-1 can reconstitute mitogenic sensitivity to insulin or IL-4 in the 32D cell line, which does not express either IRS-1 or 4PS and is insensitive to IL-4 and insulin. Mutational analysis of IRS-1 was carried out to determine the regions that are important in mediated IL-4 or insulin- induced signaling in 32D cells. Point mutation at Tyr-895 within GRB2 binding site abolished GRB2-IRS-1 interaction with little or no effect on insulin-induced mitogenicity, suggesting that essential function of IRS-1 in proliferative signaling is largely unrelated to IRS-1-GRB-2 complex formation. A PH domain deletion mutant failed to undergo insulin-induced tyrosine phosphorylation, suggesting its role in receptor coupling. The most prominent tyrosine-phosphorylated substrate induced by IL-4 was a 170 kd (p170) protein, designated 4PS (IL-4-induced tyrosine-phosphorylated substrate). 4PS was purified and cloned from murine myeloid cell line, FDC-P2. Alignment of the 4PS sequence with IRS-1 revealed two distinct functional regions: a poorly conserved COOH- terminus with multiple common and unique tyrosine phosphorylation motifs, and a highly conserved NH-2-terminus containing a pleckstrin homology (PH) domain and phosphotyrosine binding (PTH) domain that mediates receptor coupling. Introduction of 4PS cDNA into 32D cells restored their mitogenic sensitivity to IL-4 and insulin. 4PS was redesignated IRS-2 due to its functional and structural similarities with IRS-1. IL- 13 is a recently described cytokine that displays many structural biological functions similar to IL-4. IL-13 induced a potent mitogenic response and prominent tyrosine phosphorylation of p170 in IL-3-dependent TF-1 cells. A series of analyses indicated that p170 was indeed 4PS/IRS- 2, the same substrate phosphorylated by IL-4. These results may provide a basis for their overlapping biological functions. Moreover, IL-13 and IL-4 both stimulated tyrosine phosphorylation of p170 in murine L cell fibroblasts. Enhanced phosphorylation of p170 was observed in response to IL-4, but not IL-13 treatment of L cells transfected with IL-2Rg. These results indicate that IL-13, unlike IL-4, does not utilize IL-2Rg in its receptor complex.